Gram-positive bacteria deprived of HtrA protease activity and their uses

ABSTRACT

The invention concerns bacteria strains, obtained from gram-positive bacteria whereof the genome size is not more than 3.2 Mb, and wherein the HtrA surface protease is inactive. Said strains are useful for expressing exported proteins of interest.

This application is a divisional of copending U.S. application Ser. No.09/869,106 filed on Jun. 22, 2001, which is a U.S. National PhaseApplication of International Application No. PCT/FR99/03270, filed onDec. 23, 1999, which claims the benefit of French Application No.98/16462, filed Dec. 24, 1998, all of which are herein incorporated byreference in their entirety.

The invention relates to the production, in Gram-positive bacteria, ofexported proteins.

The general term “exported proteins” denotes proteins which aretransported across the cytoplasmic membrane. In the case ofGram-positive bacteria, this transport results in the secretion of theprotein into the medium, or its association with the cell surface.

One of the main problems which arises during the production of exportedproteins of interest by host bacteria lies in the degradation of theseproteins during and/or after their exportation, at the cell envelope orat the cell surface. This degradation often leads to a decrease in theyield, and/or a modification of the structure and of the activity of theprotein.

The enzymes responsible for this degradation of exported proteins arebacterial proteases, themselves exported in the envelope; they are“housekeeping” proteases, one of the main functions of which is normallya role of degradation of abnormal or incorrectly folded exportedproteins which accumulate in the medium or in the envelope, inparticular under conditions of stress, and the role of which is also therecycling of exported proteins.

Heterologous proteins, which are often incompletely recognized by thechaperone proteins involved in protein folding in the host bacterium,are particularly sensitive to attack by these proteases.

The oldest characterized exported housekeeping protease is the E. coliserine protease HtrA/DegP. It is a protease which as a periplasmiclocation, and which is expressed under the control of a promoter whichis inducible at high temperature; Beckwith and Strauch (Proc. Natl.Acad. Sci. USA 85:1576-1580, 1988) have observed that it is involved inthe proteolysis of proteins made from fusion between exported proteinsof E. coli and the PhoA exportation reporter. They have proposed theinactivation of this protease in E. coli in order to limit thedegradation of the heterologous exported proteins.

Mutant E. coli strains, in which the gene encoding the HtrA/DegPprotease has been inactivated, have thus been obtained [Beckwith andStrauch, abovementioned publication, and PCT application WO 88/05821];however, it has been noted that this inactivation results in a slowingdown of the kinetics of degradation, but is not sufficient to abolish itbecause of the existence, in the envelope, of other proteases whichdegrade the exported proteins.

In E. coli several envelope housekeeping proteases, which carry outfunctions similar to those of HtrA/DegP, have been characterized: theyare in particular the HhoA/DegQ and HhoB/DegS proteases, which arestructurally homologous to HtrA/DegP, and proteases which arestructurally different but functionally comparable (ApeA/proteaseI,OmpT, OmpP, Prc/Tsp, SppA/proteaseIV, PrtIII and SohB).

Studies relating to other bacteria have also made it possible todemonstrate the existence, in each species studied, of several exportedhousekeeping proteases. For example, a large number of bacterial specieshave several proteases of the HtrA family (Pallen and Wren, Mol.Microbiol. 19:209-21, 1997); three homologues of HtrA have beenidentified in B. subtilis (YyxA, YkdA and YvtB/Yirf), Synechocystis(HtrA, HhoA and HhoB), Pseudomonas aeruginosa and Aquifex aeolicus, twoin Hemophilus influenzae (HtoA and HhoB), Campylobacter jejuni, Brucellaabortus and Yersinia enterolitica, and four in Mycobacteriumtuberculosis. Various Gram-positive bacteria also have serine proteasesconsidered to be related to the HtrA family on the basis of homology inthe catalytic domain: EtA, EtB and V8/StsP of S. aureus, GseP ofBacillus licheniformis and Spro of Mycobacterium paratuberculosis(Koonin et al., Chap 117 in Escherichia coli and Salmonella typhimurium,2203-17, 1997). Finally, exported proteases which are not related toHtrA have also been demonstrated, for example in B. subtilis (Margot andKaramata, Microbiology, 142:3437-44, 1996; Stephenson and Harwood Appl.Environn. Microbiol. 64:2875-2881, 1998; Wu et al. J. Bacteriol.173:4952-58, 1991).

It has therefore been proposed to combine mutations affecting severalexported proteases in order to obtain an effective decrease in thedegradation of heterologous exported proteins.

For example, an E. coli strain mutated in the degP/htrA, ompT, prt andprc genes (Meerman and Georgiou, Bio/technology 12:1107-10, 1994), and aB. subtilis strain deficient in the six extracellular proteases (Wu etal., 1991, abovementioned publication), have been constructed with thisaim. However, the use of these strains does not make it possible tocompletely eliminate the proteolysis of the exported proteins. Forexample, in the case of the B. subtilis strain described by Wu et al.,although the residual extracellular protease activity is negligible(<1%), degradation of the heterologous exported proteins remainssignificant. In order to overcome this problem, that same team hascarried out further modifications to this strain in order to make itoverproduce various chaperones (Wu et al., J. Bacteriol. 180:2830-35,1998). Furthermore, although the inactivation of the gene of one ofthese exported housekeeping proteases does not have any notableconsequences for the bacterium, the accumulation of mutations may affectstrain viability; Meerman and Georgiou (1994, abovementionedpublication) thus observe a decrease in growth rate which can range upto 50%.

In lactic acid bacteria, only a few exported proteases have beenstudied; the most well characterized at the present time is the proteasenamed PrtP (Kok, FEMS Microbiol. Reviews 87:15-42, 1990), which islocated at the cell surface, where it is anchored to the peptidoglycan.This protease is present in many lactic acid bacteria, in particularLactococcus lactis, and is located on a plasmid. It contributes to thenitrogen-based nutrition of bacteria by degrading milk caseins. Othersurface proteases have been purified from two species of lactic acidbacteria, Lactobacillus delbrueckeii subsp. bulgaricus and Lactobacillushelveticus, but their function has not been determined (Stefanitsi etal., FEMS Microbiol. Lett. 128:53-8, 1995; Stefanitsi and Garel, Lett.Appl. Microbiol. 24:180-84, 1997; Yamamoto, et al., J. Biochem.114:740-45, 1993). A stress-induced gene encoding a protein which ishighly homologous to the proteases of the HtrA family has recently beenrevealed in Lactobacillus helveticus (Smeds et al., J. Bacteriol.180:6148-53, 1998). It has been observed that this gene is necessary forsurvival at high temperature; a mutant Lactobacillus helveticus strainin which the htrA gene has been inactivated by insertion of a reportergene (gusA, encoding β-glucuronidase) under the control of the htrApromoter, was constructed. The study of the expression of the gusA genein this mutant made it possible to demonstrate induction of thetranscription of this gene under the same conditions as that of the htrAgene in the wild-type strains; on the other hand, no β-glucuronidaseactivity was observed.

In previous investigations directed towards studying exported proteinsof Lactococcus lactis by studying proteins fused with the Δ_(SP)Nucexportation reporter (Poquet et al., J. Bacteriol. 180:1904-12, 1998),the team of inventors has observed significant extracellular proteolysiseven though the experiments were carried out in an L. lactis subsp.cremoris strain free of any plasmid and therefore, in particular, ofthat which carries prtP.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. FIG. 1 shows the nucleotide and amino acid sequence of the geneHtrA_(L1) (SEQ ID NO: 1). The amino acids of the catalytic domain andtransmembrane domain are box framed. The positions of primers F, G, andA are indicated by arrows.

FIGS. 2 A and B. FIGS. 2 A and B show the growth curves of thehtrA⁺/htrA strain, htrA strain, and the wild-type IL1403 strain. Thegrowth was monitored by measuring the OD₆₀₀ at the indicated timeintervals. FIG. 2A shows the growth of htrA⁺/htrA, htrA, and IL1403strains at 30° C. FIG. 2B shows the growth of htrA⁺/htrA, htrA, andIL1403 strains at 37° C.

FIG. 3. FIG. 3 shows the effect of HtrA_(L1) mutation on the stabilityof Nuc protein. The degradation profiles of Nuc protein in IL1403 (firstthree wells), htrA (three central wells) and htrA⁺/htrA (last threewells) strains are shown. The immunological detection of the Nuc proteinwas carried out on the protein samples extracted from the total culture(T), cells alone (C) and from the medium (M) of each strain.

FIG. 4. FIG. 4 shows the effect of HtrA_(L1) mutation on the stabilityof Usp-_(sp)Nuc protein. The degradation profiles of Usp-_(sp)Nucprotein in IL1403 (first three wells), htrA (three central wells) andhtrA⁺/htrA (last three wells) strains are shown. The immunologicaldetection for the Usp-_(sp)Nuc protein was carried out on the proteinsamples extracted from the total culture (T), cells alone (C) and fromthe medium (M) of each strain.

FIG. 5. FIG. 5 shows the effect of HtrA_(L1) mutation on the stabilityof N1p4-_(sp)Nuc protein. The degradation profiles of N1p4-_(sp)Nucprotein in IL1403 (first well), htrA (central well) and htrA⁺/htrA (lastwell) strains are shown.

FIG. 6. FIG. 6 shows the effect of HtrA_(L1) mutation on the stabilityof Exp5-_(sp)Nuc protein. The degradation profiles of Exp5-_(sp)Nucprotein in IL1403 (first well), htrA (central well) and htrA⁺/htrA (lastwell) strains are shown.

FIG. 7. FIG. 7 shows a zymogram of the bacteriolysin activity of AcmAprotein. The degradation profiles of AcmA protein in IL1403 (first threewells), htrA (three central wells) and htrA⁺/htrA (last three wells)strains are shown. The detection of the AcmA protein was carried out onthe protein samples extracted from the total culture (T), cells alone(C) and from the medium (M) of each strain.

The inventors undertook to investigate extracellular proteasesresponsible for this proteolysis.

They have thus discovered, in L. lactis, the existence of a gene of thehtrA family.

This gene, detected in the genome of the IL1403 strain of L. lactissubsp. lactis, encodes a 408 amino acid protein, hereinafter namedHtrA_(L1), the nucleotide sequence and the amino acid sequence of whichare represented on FIG. 1, and appear in the attached sequence listing(SEQ ID NO: 1). This protein is very homologous to E. coli HtrA, and tovarious other known members of the HtrA family, as shown in table Ibelow, which illustrates the percentages of identity and of similaritybetween HtrA_(L1) and various proteins of the HtrA family: TABLE IProtein Organism % identity % similarity HtrA/DepP/Do protease E. coli31.5 38.2 HhoA/DegQ E. coli 34.0 40.8 HhoB/DegS E. coli 29.9 37.3 HtrAS. typhimurium 32.4 39.1 HtoA H. influenzae 31.9 39.2 HhoB/DegS H.influenzae 31.2 40.0 spHtrA S. pneumoniae 55.6 62.0 HtrA Lb. helveticus46.9 54.1 YyxA B. subtilis 43.5 52.0 YkdA B. subtilis 42.5 49.4

The HtrA protein of the IL1403 strain of L. lactis subsp. lactis has thethree amino acids Ser, His and Asp, which define the catalytic sitecharacteristic of serine proteases related to trypsin, among which isthe HtrA family; in addition, it has, around these three amino acids,the following three motifs: DAYVVTNYH₁₂₇VI, D₁₅₇LAVLKIS, andGNS₂₃₉GGALINIEGQVIGIT, which correspond to the consensus regions definedby Pallen and Wren (Mol. Microbiol. 19:209-21, 1997) for the catalyticdomain of the HtrA proteases: -GY-TN-HV-, D-AV- and GNSGG-L-N-G-IGIN.

At its N-terminal end, it has a hydrophobic amino acid sequenceL₁₀LTGVVGGAIALGGSAI₂₆ corresponding to a putative transmembrane segment.The HtrA_(L1) protein of L. lactis subsp. lactis is therefore thought tobe an integral protein of the cytoplasmic membrane. According to the“positive inside” rule concerning the topology of these proteins (VonHeijne, Nature, 341:456-8, 1989), it topology corresponds to the “C-out”type, i.e. its C-terminal portion, which comprises in particular itscatalytic site, would be exposed to the outside of the plasma membrane.Like the HtrA protease of E. coli, L. lactis subsp. lactis HtrA_(L1)therefore appears to be an envelope protease which can degrade exportedproteins. The amino acids of the catalytic domain and of thetransmembrane domaine are framed on FIG. 1.

The inventors have inactivated this gene by mutation; at optimumtemperature (30° C.), the mutant L. lactis subsp. lactis strain thusobtained is viable and grows normally; on the other hand, its growth andviability are affected at higher temperatures (from 37° C.), both onplates and in liquid medium.

In addition, the inventors have studied the effect of this mutation onthe exportation of various fusion proteins, and have noted that theinactivation of the HtrA_(L1) protease in L. lactis is sufficient tocompletely abolish the degradation of the exported proteins; this effectis surprising given the residual proteolysis observed previously inother bacteria after inactivation on proteases of the HtrA family.

A subject of the present invention is a process for producing a proteinof interest, characterized in that it comprises culturing a bacterialstrain which expresses said protein of interest, and which can beobtained from a Gram-positive bacterium, the size of the genome of whichbacterium is at most equal to 3.2 Mb, preferably at most equal to 3 Mb,and advantageously at most equal to 2.5 Mb, by mutation whichinactivates the HtrA surface protease of said bacterium;

and producing said protein of interest exported by said strain.

According to a preferred embodiment of the present invention, thestarting Gram-positive bacterium is chosen from bacteria of the groupconsisting of the Streptococcaceae, and Lactobacillaceae.Advantageously, it is chosen from lactococci.

It may be also be chosen from bacteria belonging to the group consistingof the Bacillaceae, for example to the Listeria genus, and theEnterococcaceae, in particular of the Enterococcus genus.

Advantageously, said bacterial strain may also comprise one or moreother modifications of its genome, directed toward improving theproduction and/or secretion of proteins expressed in said bacterium,and/or toward avoiding their degradation. Depending on the type ofprotein intended to be produced, it is possible, for example, to use abacterial strain in which the PrtP protease activity has beeninactivated, and/or a bacterial strain which overproduces a proteinallowing the stabilization of exported proteins, such as the Nlp4protein of Lactococcus lactis, or a homologue thereof (Poquet et al.1998, abovementioned publication).

A subject of the present invention is also any bacterial strain whichcan be obtained from a Gram-positive bacterium, the size of the genomeof which bacterium is at most equal to 3.2 Mb, as defined above, bymutation which inactivates the HtrA surface protease of said bacterium,and which also comprises at least one cassette for expressing a gene ofinterest, with the exception of a Lactobacillus helveticus straincomprising a single expression cassette consisting of the sequenceencoding the gusA reporter gene inserted into the htrA gene of saidstrain, under the transcriptional control of the promoter of said gene.

The term “expression cassette” is intended to mean any recombinant DNAconstruct comprising a gene of interest, the expression of which isdesired, or a site allowing the insertion of said gene, placed under thecontrol of regulatory sequences for transcription (promoter,terminator), which are functional in the host bacterium underconsideration.

For the purpose of the present invention, the term “HtrA protease” isintended to mean any serine protease of the trypsin type, havingfunctional and structural similarities with the HtrA protease of E. coliwhich are sufficient for it to be included in the same family, i.e.:

a catalytic site formed by the three amino acids Ser, His and Asp;

the presence, around this catalytic site, of the consensus regions:-GY-TN-HV-, D-AV- and GNSGG-L-N-G-IGIN;

an exportation signal enabling the protease to be transported to thecell surface of the bacterium, (it may, for example, be a signalpeptide, a transmembrane domain, a signal for anchorage to the wall,etc.).

In order to implement the present invention, mutant bacteria lackiingHtrA activity can be produced by carrying out one or more mutations, inparticular in the sequence encoding the HtrA protease and/or in theregulatory sequences allowing the expression of the htrA gene, so as toprevent the expression of a functional HtrA protease. These mutationscan be carried out conventionally, by deletion, insertion or replacementof at least one nucleotide or one nucleotide sequence in the htrA gene;they can result either in the absence of production of HtrA, or in theproduction of an HtrA protease in which at least one amino acid requiredfor activity has been deleted or replaced.

The suitable mutagenesis techniques are known per se; advantageously,use will be made of site-directed mutagenesis techniques, since the dataavailable on the proteases of the HtrA family make it possible, eventhough more precise information on the specific sequence of the genewhose inactivation is desired is not available, to target themutation(s) on conserved domains which are required for activity (forexample the catalytic domain).

The present invention can be implemented in many domains.

Firstly, it can be used in the domain of the production of proteins ofinterest (for example enzymes, human proteins, etc.) by geneticengineering, using cultures of bacteria transformed with a gene ofinterest. In this domain, the present invention makes it possible toimprove the yield of exported proteins (and in particular secretedproteins), and to avoid their contamination with inactive proteolyticproducts: this makes it possible to purify them easily and lessexpensively.

For this application, use will preferably be made of the mutant strainsproduced from nonpathogenic bacteria, such as Lactococcus spp. orLactobacillus spp., and also food streptococci, Streptococcusthermophilus.

The mutant strains produced from bacteria conventionally used in theagro-foods industry, such as lactic acid bacteria (in particularlactococci, lactobacilli and thermophilic streptococci), canadvantageously be used in this domain. For example, they can be used inthe composition of ferments, in order to produce heterologous proteinsmaking it possible to improve the quality of the finished fermentedproduct; thus, the exportation of foreign enzymes produced by a mutantL. lactis strain in accordance with the invention, within cheesesfermented with L. lactis, may improve their maturing and theirorganoleptic qualities.

These mutant strains can also be used for producing dietetic products ormedicinal products. In this domain, mutant strains in accordance withthe invention can, for example, be used in order to express, prior tothe ingestion of the product and/or after its ingestion, proteins with aprophylactic or therapeutic effect, such as enzymes (for facilitatingdigestion, for example), proteins for stimulating the immune system,immunization antigens, etc. In most cases, for use in this domain, andin order to guarantee maximum innocuity, mutant strains produced fromnonpathogenic bacteria and, advantageously, from bacteria conventionallyused for food will be preferred. However, in the context of uses forimmunization, mutant strains produced from pathogenic bacteria (inparticular streptococci, staphylococci, enterococci or listeria), andpreferably from variants of these bacteria already carrying one or moremutations which attenuate their pathogenic power, can be used; theinactivation of the HtrA protein, in limiting the capacities of survivalof these bacteria under conditions of stress, may contribute toattenuating their virulence, as previously observed in the case ofcertain Gram-negative bacteria.

In the context of certain applications, in which the host bacterium mustbe viable and capable of producing proteins at temperatures of about 35to 40° C., for example the production, in a fermentor, of certainproteins, or the production, after ingestion, in the digestive tract ofhumans or animals, of proteins with therapeutic or prophylacticactivity, mutant strains produced from thermophilic bacteria, such asStreptococcus thermophilus, will advantageously be used.

The present invention will be more clearly understood with the aid ofthe continuation of the description which follows, which refers tononlimiting examples illustrating the production of L. lactis mutants inwhich the HtrA surface protease is inactive, and the properties of thesemutants.

EXAMPLE 1 Inactivation of the hrtA Gene of L. lactis

htrA gene, carried by the chromosome of the IL1403 strain (Chopin et al.Plasmid, 11, 260-263, 1984) of L. lactis subsp. lactis, was inactivatedby integration of a suicide plasmid carrying a 665 bp internal fragmentof the gene (FA).

As a positive control for integration, a suicide plasmid carrying a 902bp fragment truncated in the 3′ region (GA), the integration of whichonto the chromosome restores a wild-type copy of the gene, was used.

These fragments were obtained beforehand by PCR amplification from thegenomic DNA of the IL1403 strain of L. lactis subsp. lactis, using thepairs of primers F/A and G/A: - F[5′-GGAGCCA(G/T) (A/C/T)GC(A/G/C/T)(C/T)T (A/G/T)GG-3′]

located downstream of the ATG initiation codon -G[5′-GTTTCCACTTTTCTGTGG-3′]

located upstream of the htrA promoter -A[5′-TT(A/T)CC(A/T)GG(A/G)TT(A/G/T)AT(A/G/C/T) GC-3′]located upstream of the serine codon of the catalytic site.The positioning of the F, G and A primers is indicated on FIG. 1.

The amplification was carried out under the following conditions:

reaction mixture: 0.2 mM of each dNTP, 5 μM of each oligonucleotide,approximately 500 ng of chromosomal DNA, 2 mM of MgCl₂ and 1.25 units ofTaq-DNA-pol (Boehringer Mannheim), in the Taq buffer provided by themanufacturer;

temperature conditions: 5 min 94° C., 30 cycles (30 sec at 94° C., 30sec at 46° C., and 30 sec at 72° C.), and 4° C.

The amplified fragments were ligated to the linear pGEM^(T) plasmid(Promega). After transformation of E. coli TG1 with the ligationproducts, the clones which are resistant to ampicillin and lackβ-galactosidase activity are selected. The plasmids obtained, bearingthe FA and GA fragments, respectively, are named pES1.1 and pES2.1.

The FA and GA inserts were subcloned into a suicide vector carrying achloramphenicol resistance gene. Since this vector is incapable ofreplicating alone in the absence of the RepA protein which is requiredfor initiating its replication, co-integrants were created by ligationbetween each of the pES1.1 and pES2.1 plasmids and the suicide vector,linearized beforehand.

After transformation of the E. coli TG1 strain, and selection of thechloramphenical-resistant clones, the pGEM^(T) portion of theco-integrants was deleted and the vectors were re-circularized. Theplasmids obtained are multiplied in the TG1 repA⁺ strain of E. coli;after selection of the chloramphenicol-resistant clones, the suicideplasmids named pVS6.1 and pVS7.4 are obtained.

pVS6.1 contains the FA fragment, and pVS7.4 contains the GA fragment, ofthe htrA_(L1) gene of the IL1403 strain of L. lactis subsp. lactis.

These plasmids were used to transform the IL1403 strain of L. lactissubsp. lactis; the clones which had integrated these plasmids at thehtrA locus on the chromosome were selected in the presence ofchloramphenicol.

In both cases, several independent chloramphenicol-resistant clones wereobtained. Five clones of each class termed A to E in the case of theintegration of pVS6.1, and 17 to 22 in the case of the integration ofpVS7.4, were chosen for analysis.

For each of these clones, the integration at the htrA locus wasconfirmed by Southern transfer.

Two clones, A and 17, were chosen for the following analyses; theyconstitute the two prototypes of the mutant strains, which hereinafterwill be named:

htrA (null mutation of the htrA_(L1) gene, Cm^(R)); this strain does notexpress any active HtrA protease;

htrA⁺/htrA (wild-type copy+truncated copy of the htrA_(L1) gene,Cm^(R)); this strain expresses an active Htra_(L1) protease.

EXAMPLE 2 Role of the htrA_(L1) Gene of L. lactis in Survival at HighTemperature

The two strains htrA and htrA⁺/htrA are cultured, in liquid culture,under the conventional conditions for growth of L. lactis, i.e. at 30°C. and in the presence of oxygen, but without stirring, and in thepresence of chloramphenicol.

The behavior of the htrA strain of L. lactis subsp. lactis at 30° C. andat 37° C. was studied using the htrA⁺/htrA strain and also the IL403parent-strain (cultured in the absence of chloramphenicol) as control.

The bacteria were cultured overnight at room temperature, in an M17medium containing 1% of glucose (+2.5 μg/ml of chloramphenicol for boththe htrA strain and the htrA⁺/htrA strain). The cultures were diluted100-fold in the morning, in the same medium, and divided into twobatches placed in semi-anaerobiosis at 30° C. or at 37° C. The growthwas monitored by measuring the OD₆₀₀.

The results are illustrated in FIG. 2.

At 30° C. (FIG. 2A), it is noted that the htrA⁺/htrA strain (▪), thehtrA strain (♦), and the wild-type IL1403 strain (♦) have very closegeneration times: 65 min for the wild-type strain, 70 min for htrA⁺/htrAand 75 min for htrA; finally, for the 3 cultures, the OD₆₀₀ valuescorresponding to the stationary phase are very comparable (OD₆₀₀=2.1 to2.2).

These results indicate that there is no significant difference in growthbetween these three strains at 30° C.

At 37° C. (FIG. 2B), the htrA⁺/htrA strain (▪) has a generation time of100 min and the OD₆₀₀ of the stationary phase is less than at 30° C.(OD₆₀₀=1.25). Less growth at 37° C. than at 30° C. is also observed forthe wild-type IL1403 strain (♦); the generation time is 65 min, but theOD₆₀₀ of the stationary phase is less than at 30° C. (OD₆₀₀=1.9). In thecase of the htrA strain (♦), the growth is very slight, or even zero,and the OD₆₀₀ does not exceed 0.1, even after culturing for 7 h.

It emerges from these results that the htrA strain of L. lactis subsp.lactis is heat-sensitive and that the htrA mutation is lethal at 37° C.

EXAMPLE 3 Role of the htrA_(L1) Gene of L. LACTIS in Surface Proteolysis

The effect of the htrA_(L1) mutation on the stability of five exportedproteins was tested. These proteins are:

i) a heterologous protein, the secreted nuclease of S. aureus, Nuc; thisprotein is expressed by the plasmid pNuc3 (Le Loir et al., J. Bacteriol.176:5135-5139, 1994; Le Loir et al., J. Bacteriol. 180:1895-903, 1998);

ii) three hybrid proteins (Usp-Δ_(SP)Nuc, Nlp4-Δ_(SP)Nuc andExp5-Δ_(SP)Nuc) resulting from the fusion between the Δ_(SP)Nuc reporterand fragments of exported proteins of L. lactis: the secreted proteinUsp45 (Van Asseldonk et al., Gene 95:155-60, 1990), the lipoprotein Nlp4and the protein Exp5 (which is, itself, a protein made from fusionbetween an exported protein and a cytoplasmic protein); these proteins,and also the plasmids pVE8009, pVE8024 and pVE8021 which express them,respectively, are described by Poquet et al. (1998, abovementionedpublication);

iii) a naturally exported protein of L. lactis, AcmA.

In the wild-type MG1363 strain of L. lactis subsp. cremoris,Usp-Δ_(SP)Nuc is secreted and Nlp4-Δ_(SP)Nuc is associated with thecells; for these two proteins, various degradation products, among whichthe NucA peptide originating from the Δ_(SP)Nuc portion of the fusion,are detected in the medium, along with the mature form; with regard tothe Exp5-Δ_(SP)Nuc tripartite fusion, it is very unstable and the matureform is not detected in the medium, only the degradation products,including the NucA peptide. The mature form, and also the degradationproducts of these three hybrid proteins, can be detected using anti-NucAantibodies.

The naturally exported protein of L. lactis chosen is the bacteriolysinAcmA (Buist et al., J. Bacteriol. 177:1554-1563, 1995). This protein,which degrades peptidoglycan, is both secreted and associated with thesurface, probably by affinity with its substrate. It provides, both inthe MG1363 strain of L. lactis subsp. cremoris and the IL1403 strain ofL. lactis subsp. lactis, proteolysis products which are active andtherefore detectable, like the intact protein, by zymogram.

The strains transformed with the plasmids expressing these variousproteins are cultured at 30° C. for several hours, at least up to themiddle of the exponential phase or up to the start of the stationaryphase.

For each plasmid, cultures of the three strains IL1403, hrtA andhtrA⁺/htrA, which had reached comparable OD₆₀₀ values, were used toextract protein samples: a) from the total culture, b) from the cellsand c) from the medium, according to the protocol described by Poquet etal. (1998, abovementioned publication).

These samples are subjected to electrophoresis (SDS-PAGE) on denaturinggel.

In order to detect the Nuc, USP-Δ_(SP)Nuc, Nlp4-Δ_(SP)Nuc andExp5-Δ_(SP)Nuc proteins and their degradation products, the proteins aretransferred onto a membrane, followed by immunological revelation usinganti-NucA antibodies, which are detected using a protein G/peroxidaseconjugate (BIO-RAD) and a chemiluminescence kit (Dupont-Nen).

AcmA is detected by zymogram (Buist et al., 1995, abovementionedpublication): micrococci, in which the wall is sensitive to AcmA, areincluded in the electrophoresis gel at the concentration of 0.2%, whichmakes it opaque; after electrophoresis, the gel is treated at 37° C.overnight in a buffer containing 50 mM of Tris/HCl at pH 7 and 0.1% ofTriton X100, which allows lysis of the micrococci by AcmA or its activeproteolytic products. The gel is then colored with methylene blue at0.1% in 0.01% KOH: the bands corresponding to the AcmA activity appearas transparent hydrolysis halos on a blue background.

For each protein, the degradation profiles in the IL1403, htrA andhtrA⁺/htrA strains were compared by observing the protein contentaccumulated during culturing for several hours.

FIGS. 3 to 6 show, respectively, the results of immunological detectionfor the Nuc, Usp-Δ_(SP)Nuc, Nlp4-Δ_(SP)Nuc and Exp5-Δ_(SP)Nuc proteins.For the Nuc (FIG. 3) and Usp-Δ_(SP)Nuc (FIG. 4) proteins, [lacuna]

FIG. 7 represents a zymogram of the bacteriolysin activity of AcmA; thedetection was carried out on the total culture (T), the cells alone (C)or the medium (M).

In the IL1403 Strain:

For the secreted proteins Nuc and Usp-Δ_(SP)Nuc (FIGS. 3 and 4: firstthree wells), and for the lipoprotein Nlp4-Δ_(SP)Nuc (FIG. 5: firstwell), a three-band profile is detected, as previously observed in theMG1363 strain (Le Loir et al., 1994; Poquet et al., 1998, abovementionedpublications):

a) the band with the highest molecular weight is the precursor fromwhich the signal peptide has not been cleaved, which is confirmed by itspresence exclusively in the cells (FIGS. 3 and 4);

b) the intermediate band is the mature form after cleavage of the signalpeptide, and, in the case of the secreted proteins Nuc and Usp-Δ_(SP)Nuc(FIGS. 3 and 4), it is present exclusively in the medium;

c) the band with the lowest molecular weight is the NucA peptide whichpractically comigrates with the commercial NucA form purified from S.aureus (the slight difference in migration being due to the differentcleavage specificities in S. aureus and L. lactis), and which is bothreleased into the medium and associated with the cells.

For the Exp5-Δ_(SP)Nuc protein (FIG. 6: first well), two forms aredetected only with great difficulty, one having a high molecular weightand one having a low molecular weight, NucA, which practicallycomigrates with the purified commercial form; there is, therefore,practically total proteolysis in IL1403.

For the AcmA protein (FIG. 7: the first three wells), a four-bandprofile, as previously observed in the MG1363 strain (Buist et al.,1995, abovementioned publication), is detected:

a) the band with the highest molecular weight is the precursor fromwhich the signal peptide has not been cleaved, which is presentexclusively in the cells;

b) the band with a slightly lower molecular weight is the mature formafter cleavage of the signal peptide, which is both secreted into themedium and associated with the surface of the cells by affinity for itssubstrate;

c and d) the two bands of lower molecular weight are active proteolyticproducts, both secreted into the medium and associated with the surfaceof the cells by affinity for their substrate.

In the htrA⁺/htrA Strain:

(FIGS. 3 and 4: last three wells, FIGS. 5 and 6: last well, and FIG. 7:last three wells). The profiles observed are absolutely identical tothose observed in the wild-type strain. The htrA⁺/htrA strain thereforeexhibits a wild-type proteolytic phenotype which is explained by thewild-type copy of the htrA_(L1) gene which it possesses.

In the htrA Strain:

(FIGS. 3 and 4: three central wells, FIGS. 5 and 6: central well, andFIG. 7: three central wells).

In all cases, none of the proteolytic products are detected;simultaneously, the amount of mature protein (or of high molecularweight protein in the case of Exp5-Δ_(SP)Nuc) increases.

These results show that the product of the htrA_(L1) gene is clearlyresponsible for the degradation of the secreted proteins, and that itsinactivation leads to the complete abolition of this degradation.

1-10. (canceled)
 11. A method of producing a fermented product,comprising: culturing a bacterial strain with a fermentation substrateunder conditions suitable to produce a fermented product, wherein thebacterial strain does not express a functional HtrA.
 12. A method ofproducing a dietetic food, comprising: culturing a bacterial strain witha substrate under conditions suitable to produce a dietetic food,wherein the bacterial strain does not express a functional HtrA.
 13. Amethod of producing a medicinal product, comprising: culturing abacterial strain with a suitable substrate under conditions effective toproduce a medicinal product, wherein the bacterial strain does notexpress a functional HtrA.
 14. A method according to claim 13, whereinthe medicinal product is a vaccine.